Enoxacin and Binhib16 were recently identified as novel inhibitors of osteoclast formation and function in in vitro assays. Previous studies had indicated that inhibitors of an interaction between the B-subunit of vacuolar H+ATPase (V-ATPase) and actin filaments should block the ability of osteoclasts to resorb bone. A computer based screen for small molecules predicted to bind the actin binding surface of subunit B and inhibit its interaction with actin filaments identified enoxacin and Binhib16. These molecules were then shown to block binding of pure rabbit muscle actin filaments to B-subunit in the test tube. Enoxacin and Binhib16 were tested for effects on osteoclast formation and function in vitro in calcitriol-stimulated mouse marrow cultures. Both blocked osteoclast formation and the function of pre-differentiated osteoclasts at a concentration of approximately 10 <M. In contrast, these molecules did not have detectable effects on the number of osteoblasts produced in marrow cultures, as judged by the number of alkaline phosphatase positive cells, or on MC3T3-E1 osteoblasts. These data suggest that enoxacin and Binhib16 might inhibit osteoclasts by novel mechanisms, without having inhibitory effects on osteoblasts. The overall goal of this proposal is to perform preliminary tests to determine whether enoxacin and Binhib16 hold promise as agents for use in the treatment of various dental applications in which inhibition of osteoclastic bone resorption would be beneficial. The following central hypothesis is proposed: Enoxacin and Binhib16 are the first examples of a new class of therapeutic agents for the inhibition of osteoclastic bone resorption by targeting V-ATPase binding to the actin cytoskeleton. Four specific aims are suggested. In Aim 1, we will develop a method for sustained local release of enoxacin and Binhib16 making use of Elvax. Numerous studies have utilized Elvax for similar applications. In Aim 2, enoxacin and Binhib16 will be tested for its capacity to reduce periodontal bone loss in a well-established rat model of periodontitis. Aim 3 will test whether treatment with enoxacin and Binhib16 reduces resorption associated with re- implantation making use of an established ferret system. Aim 4 will test the capacity of enoxacin and Binhib16 to block orthodontic tooth movement in a rat model. This could provide a novel means for providing orthodontic anchorage. This study should provide initial evidence regarding the potential of inhibitors of V-ATPase-actin interactions for use in dental procedures. Support for the current studies is necessary in order to provide sufficient preliminary data to determine whether large scale pre-clinical studies are justified. Given that the proposed research represents a fresh and innovative approach to the use of bioactive small molecules for dental applications, and could have a large clinical impact, we believe that NIH support in the form of an R21 grant is warranted. PUBLIC HEALTH RELEVANCE: A bioactive agent that inhibits osteoclastic bone resorption would be useful for various dental applications. Novel small molecule inhibitors of osteoclast function, enoxacin and Binhib16, have been identified using a supercomputer-based assay. The current proposal will conduct pilot preclinical tests of these molecules in animal models to examine their suitability for periodontic, endodontic and orthodontic uses.